The aromatase enzyme (CYP19A1) is required for the biosynthesis of estradiol, a steroid needed for normal follicular and luteal cell function and essential for reproduction. Aromatase expression in ovarian follicular cells is stimulated mainly by the activation of a cAMP response element-like sequence (CLS) found in the proximal promoter of the cyp19 gene. We have shown that this promoter also drives aromatase expression in luteal cells;however, in these cells neither cAMP nor CLS are involved in the expression of this enzyme. This suggests that alternative mechanisms control estradiol production in the corpus luteum. On the other hand, we have demonstrated that aromatase expression in corpora lutea of pregnant rats correlates with changes in the binding of luteal nuclear proteins to an activation protein 3 response element (AP3-RE) found in the proximal promoter. Mutation of the AP3-RE, but not of the CLS binding site, abolishes aromatase promoter activity in luteal cells. Based on this evidence, we hypothesize that the AP3-RE plays a key role in the regulation of estradiol production in luteal cells. We have been unable to test this hypothesis because the identity of the protein that binds to AP3-RE is unknown. The aim of this exploratory R21 application is to identify luteal proteins that specifically bind to the AP3-RE. Two approaches will be used: i) mass spectrometry analysis of affinity-column purified proteins and ii) screening of a luteal cDNA expression library. Molecular biology, protein chemistry, and genetic techniques are currently being used in my laboratory or are available to me at the Proteomics and Informatics Services Facility at UIC. Once the AP3-RE binding protein has been identified, recombinant GST fusion proteins will be produced. Purified proteins will be used to develop specific antibodies. Activation protein 3 expression vectors and antisense probes will be used to investigate the role of this protein in aromatase regulation and luteal gene expression. This project will enhance our understanding of the molecular mechanisms that govern estradiol production. PUBLIC HEALTH RELEVANCE: The aim of this proposal is to identify proteins that bind to the promoter of the aromatase gene. This gene encodes an enzyme that catalyzes the synthesis of estradiol. Estradiol is an essential hormone in reproduction and crucial for the normal function of the brain, liver, and bone. Estradiol also stimulates the proliferation of cancer cells. Therefore, this project will have a broad impact on our understanding of the molecular mechanisms that control estradiol production under normal and pathological conditions.